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WIREs Forensic Sci

Exploring new short tandem repeat markers for DNA mixture deconvolution

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Abstract Relying on their polymorphic nature, short tandem repeats (STRs) have been extensively studied and routinely utilized as human identity markers in forensic genetics. However, even the most comprehensive STR multiplexes in use today are limited in their ability to determine the number and appropriation of component contributor alleles in DNA mixtures. The difficulty in parsing out individuals in DNA mixtures is a consequence of overlapping length‐based alleles genotyped using the polymerase chain reaction (PCR) coupled with capillary electrophoresis (CE). Many challenges exist in the resolution of minor alleles (i.e., the alleles originating from a minor contributor) from stutter and stochastic effects (e.g., inherent heterozygote peak imbalance, undetected alleles [drop out]) in a given DNA profile, in addition to the complex statistical models and algorithms necessary to render DNA mixtures interpretable from a forensic casework standpoint. Therefore, we can either adapt to complex interpretation methods, or pursue new bench science approaches to DNA mixture deconvolution. One promising area of research includes the incorporation of additional highly polymorphic STR loci to compliment current marker multiplexes and offer great potential to improve the forensic genetic analysis of DNA mixtures. This article is categorized under: Forensic Biology > Forensic DNA Technologies Forensic Biology > Interpretation of Biological Evidence Forensic Biology > Ethical and Social Implications
ForenSeq UAS (Universal Analysis Software) output of a mock mixed DNA sample of two related individuals (image taken from in‐house data using the ForenSeq DNA Signature Prep Kit with the MiSeq FGx System [Verogen, San Diego, CA, USA]). On the left, many loci in gray boxes reflect allele sharing for both length and sequence, whereas loci in orange boxes reflect instances where multiple alleles were detected, or other interpretation challenges were present (i.e., allele imbalance, missing data, etc.). On the right, the D19S433 locus is highlighted to demonstrate the true alleles (in blue), where no allele sharing between the individuals is observed, and the contribution of each component contributor in the sample can be quantified. Further, stutter and noise sequences (denoted in brown and gray bands at each allele) are also reported and quantified
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A hypothetical example of Melissa and Marie, two unrelated individuals who are contributing to a mixed length‐based genotype [14, 15] at a hypothetical locus. The resolution power from a polymerase chain reaction–capillary electrophoresis standpoint can be challenging, with many possibilities to explain the data (left image). Using MPS, and assuming at least one sequence variant in either or both of the alleles, the number of possibilities to explain the data can be reduced, and the confidence in the interpretation can be impacted (right image)
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A single nucleotide polymorphism (SNP) variant present in a homozygous length‐based [16, 16] genotype resulting in a heterozygous sequence‐based genotype (image taken from in‐house data using the ForenSeq DNA Signature Prep Kit with the MiSeq FGx System [Verogen, San Diego, CA, USA]). In the top box that depicts the string sequence for each allele, the SNPs are highlighted in green (allele 1) and red (allele 2), where dark purple nucleotides represent the repeat region of the DNA sequence whereas light purple nucleotides represent the flanking region of the DNA sequence. In the bottom left image, the SNP variation is denoted by a color change (brown and blue) whereas in the bottom right image, the SNP variation is only denoted in the string sequence above the mock electropherogram
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Example of the D9S1122 locus alignment data for nominal length‐based 12 alleles to the GRCh38 human reference genome, that highlights both repeat region (purple arrow) and flanking region (orange arrow) sequence variation. Data taken from Novroski, King, Churchill, Seah, & Budowle, 2016
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Simulation of a three locus electropherogram with an unresolvable mixture
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Simulation of a three locus electropherogram with a two‐person resolvable mixture
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Summary data from 2008 NIST Presentation by A.M. Gross at the American Academy of Forensic Sciences 60th Annual Meeting. Of the 3,106 total samples examined, 43% of samples were determined to be either 2, 3, or 4+ person mixtures. The number of contributors are divided into three categories, where blue are sexual assaults; orange are major crime (e.g., homicide, assault with weapon); and gray are high volume (e.g., property crimes, burglaries, theft) (Gross, 2008)
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A comparison of two DNA profiles comprising the same five short tandem repeats from unrelated individuals, generated using the Applied Biosystems Globalfiler polymerase chain reaction Amplification Kit (Thermo Fisher Scientific) on the Applied Biosystems SeqStudio Genetic Analyzer (Thermo Fisher Scientific). At each locus, the two individuals under comparison could share zero, one or two alleles and individuals can present as homozygous (single peak) or heterozygous (two peaks)
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Simulation of drop in and dropout for a three‐locus DNA profile. The DNA profile on the left is unaffected by drop‐in and dropout and represents the “true” alleles for each locus, or the correct genotypes for each locus. The DNA profile on the right has drop‐in visible at Locus A, and dropout visible at Locus C, in turn rendering interpretation of the DNA profile more challenging
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Electropherogram and graphical representation of size separation range limitation. Each dye channel has been optimized to contain the largest number of loci possible, taking into account length‐based allele spread at each locus. Each locus in a dye channel does not share the same size alleles with any other locus in the same dye channel. Selectively designing short tandem repeat kits that arrange loci in particular dye channels ensures that all alleles in the dye channel can be properly assigned to their respective loci. Loci with alleles of similar size are placed in different dye channels so that each locus can be genotyped correctly. Selfgenerated image using the 6‐dye chemistry of the Applied BiosystemsTM GlobalFilerTM polymerase chain reaction Amplification Kit (Thermo Fisher Scientific, Waltham, MA, USA) coupled with the Applied BiosystemsTM SeqStudioTM Genetic Analyzer System (Thermo Fisher Scientific, Waltham, MA, USA)
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An example random match probability (RMP) calculation (simplified) for two unrelated individuals at three independent loci
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A visual representation of length versus sequence variability at an arbitrary short tandem repeat locus X, where the sequence variability is not captured using traditional polymerase chain reaction–capillary electrophoresis methods
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Visual representation of basic short tandem repeat (STR) variability. (a) STRs exist as repeating elements in the genome and vary in their overall repeat length, where the underlined nucleotides denote the repeat motif in the nucleotide string; (b) STRs are comprised of different sequences and typically exist as simple, compound, or complex motifs
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Forensic Biology > Ethical and Social Implications
Forensic Biology > Interpretation of Biological Evidence
Forensic Biology > Forensic DNA Technologies

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